Although the actin cytoskeleton is vital for carcinogenesis and subsequent pathology, no microfilament-directed agent has been approved for cancer chemotherapy. One of the most studied classes of microfilament-directed agents has been the cytochalasins, mycotoxins known to disrupt the formation of actin polymers.
Trang 1R E S E A R C H A R T I C L E Open Access
Effects of cytochalasin congeners,
microtubule-directed agents, and doxorubicin
alone or in combination against human
ovarian carcinoma cell lines in vitro
Matthew Trendowski*, Timothy D Christen, Christopher Acquafondata and Thomas P Fondy
Abstract
Background: Although the actin cytoskeleton is vital for carcinogenesis and subsequent pathology, no
microfilament-directed agent has been approved for cancer chemotherapy One of the most studied classes of microfilament-directed agents has been the cytochalasins, mycotoxins known to disrupt the formation of actin polymers In the present study, we sought to determine the effects of cytochalasin congeners toward human drug sensitive and multidrug resistant cell lines
Methods: SKOV3 human ovarian carcinoma and several multidrug resistant derivatives were tested for sensitivity against a panel of nine cytochalasin congeners, as well as three clinically approved chemotherapeutic agents (doxorubicin, paclitaxel, and vinblastine) In addition, verapamil, a calcium ion channel blocker known to reverse P-glycoprotein (P-gp) mediated drug resistance, was used in combination with multiple cytochalasin congeners to determine whether drug sensitivity could be increased
Results: While multidrug resistant SKVLB1 had increased drug tolerance (was more resistant) to most cytochalasin congeners in comparison to drug sensitive SKOV3, the level of resistance was 10 to 1000-fold less for the cytochalasins than for any of the clinically approved agents While cytochalasins did not appear to alter the expression of ATP binding cassette (ABC) transporters, several cytochalasins appeared to inhibit the activity of ABC transporter-mediated efflux of rhodamine 123 (Rh123), suggesting that these congeners do have affinity for drug efflux pumps Cytochalasins also appeared to significantly decrease the F/G-actin ratio in both drug sensitive and drug resistant cells, indicative of marked microfilament inhibition The cytotoxicity of most cytochalasin congeners could be increased with the addition
of verapamil, and the drug sensitivity of resistant SKVLB1 to the clinically approved antineoplastic agents could be increased with the addition of cytochalasins As assessed by isobolographic analysis and Chou-Talalay statistics,
cytochalasin B and 21,22-dihydrocytochalasin B (DiHCB) demonstrated notable synergy with doxorubicin and
paclitaxel, warranting further investigation in a tumor-bearing mammalian model
Conclusion: Cytochalasins appear to inhibit the activity of P-gp and potentially other ABC transporters, and may have novel activity against multidrug resistant neoplastic cells that overexpress drug efflux proteins
* Correspondence: mrtrendo@syr.edu
Department of Biology, Syracuse University, 107 College Place, Syracuse, NY
13244, USA
© 2015 Trendowski et al Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made The Creative Commons Public Domain Dedication waiver
Trang 2Cytochalasins are mycotoxins known to disrupt the
for-mation of filamentous (F)-actin, thereby preventing the
formation of functional microfilaments These congeners
are characterized by a highly substituted
perhydro-isoindolone structure that is typically attached to a
macro-cyclic ring [1] More than 60 different cytochalasins from
several species of fungi have been classified into various
subgroups based on the size of the macrocyclic ring and
the substituent of the perhydroisoindolyl-1-one residue at
the C-3 position [2]; structures of representative
cytocha-lasins are shown in Fig 1 While most of our previous
work has focused on cytochalasin B, there are many other
congeners with similar activity toward microfilaments As
microfilament-disrupting agents, cytochalasins alter cell
motility, adherence, secretion, drug efflux, deformability,
morphology, and size, among many other cell properties
critical to neoplastic cell pathology [1, 2] In addition, two
of the congeners (cytochalasins B and D) have shown
par-tial specificity against neoplastic cells [3–10], consistent
with the substantial differences known to exist between
the microfilament biochemistry of neoplastic and normal
cells [11, 12] These differences in microfilament structure
may be related to key neoplastic characteristics, including
altered adherence, anchorage independent growth,
inva-siveness, and altered plasma membrane cytoskeletal
inter-actions involving expression of oncoproteins [12, 13]
Previously, we have demonstrated that cytochalasin B and its reduced congener 21,22-dihydrocytochalasin B (DiHCB) are able to sensitize multidrug resistant P388/ ADR murine leukemia cells to doxorubicin, with both con-geners showing considerable drug synergy with the nucleic acid-directed agent [14] In addition, prior research has indicated that cytochalasin B efflux is less affected by over-expression of ATP binding cassette (ABC) transporters than other cytotoxic drug classes (vinca alkaloids and anthracyclines) [15] that often exhibit drug resistance in the clinical setting Based on these observations, it appears that cytochalasin B and potentially other cytochalasin congeners might be active against multidrug resistant neoplastic cells and might also be able to overcome resistance to other cytotoxic agents currently used in the clinical setting Therefore, this study seeks to determine the effects of cyto-chalasin congeners toward drug sensitive and multidrug resistant human cancer cell lines
SKOV3 is a human ovarian carcinoma cell line frequently used in vitro and also in vivo as a xenograft in immunosup-pressed mice SKOV3 cells have slight, but noticeable resist-ance to tumor necrosis factor, as well as to cisplatin and doxorubicin [16, 17] The inherent drug resistance of SKOV3 cells can be dramatically elevated through progres-sively increasing exposure to either vinblastine or vincris-tine [18] One of the most notable multidrug resistant cell lines, SKVLB1, is 2,000-fold more resistant to vinblastine,
Fig 1 Molecular structure of the cytochalasin macrocycle and several congeners The macrocycle skeleton of cytochalasins is provided to indicate the numbering system used for these congeners In addition, the structure of 21,22-dihydrocytochalasin B is shown to indicate the differences in structure it has with the γ-lactone derivative The α, β-unsaturated ketones of cytochalasin A runs from C-20 to C-23
Trang 310,000-fold more resistant to vincristine, 260-fold more
resistant to doxorubicin, and 510-fold more resistant to
the non-clinically approved colchicine when compared to
the parental cell line [18] Further, this increase in drug
resistance is mirrored by increasing overexpression of
P-glycoprotein (P-gp), a known ABC transporter [19, 20]
Since cytochalasin B efflux is notably resistant to P-gp
overexpression, drug sensitive and drug resistant SK
human ovarian carcinoma cell lines are ideal models to
examine cytochalasin sensitivity in multidrug resistant
cancers These cell lines can also reveal potential drug
synergism when cytochalasin congeners are combined
with chemotherapeutic agents, or with calcium ion
chan-nel blockers known to inhibit P-gp drug efflux [20–24]
Methods
Preparation of human ovarian carcinoma cell lines
SK human ovarian carcinoma cell lines with varying levels
of drug resistance were provided courtesy of Dr Victor
Ling (University of British Columbia, Canada) The level
of drug resistance of the cell lines from lowest to highest
is as follows: SKOV3 (parental cell line), SKVCR0.015,
SKVCR0.1, and SKVLB1 (see Reference [18] for details on
how drug resistance is acquired) All cell lines were seeded
sus-pended in 9 ml of RPMI 1640 complete medium
contain-ing 10 % newborn calf serum (GIBCO, Grand Island, NY,
37 °C During subculture, cells were trypsinized with
0.05 % trypsin-EDTA solution 1X (Sigma-Aldrich Corp., St
Louis, MO, USA) for 5 min at 37 °C, dislodged by a sharp
knocking of the flasks during that period, washed, diluted
were seeded into 25 cm2culture flasks (4 × 104cells/cm2)
Cytochalasin B preparation
Cytochalasin B was prepared from mold mattes of
described [13, 14, 25], and purified by preparative thin
layer chromatography to greater than 99 % homogeneity
after recrystallization from chloroform Cytochalasin B
and other cytochalasins prepared in our laboratory were
shown), and were compared to commercially acquired
samples (Sigma-Aldrich Corp.) to ensure that the
iso-lated products were of a suitable grade
21, 22-Dihydrocytochalasin B Preparation
DiHCB was prepared by sodium borohydride reduction of
cytochalasin B in methanol at 25 °C as previously described
[14, 26] The product was recovered as a
chloroform-soluble fraction and crystallized from benzene:hexane
DiHCB was compared to a commercially purchased
sample of DiHCB (Sigma-Aldrich Corp.) and cytocha-lasin B (Sigma-Aldrich Corp.) using reverse phase thin layer chromatography
Cytochalasin D preparation Cytochalasin D was prepared from mold mattes of
described [14, 26], and purified by preparative thin layer chromatography to greater than 99 % homogen-eity after recrystallization from chloroform
Cytochalasin C preparation Cytochalasin C was prepared through an isomerization reaction of cytochalasin D using a Pd/charcoal catalyst at
25 °C as previously described [26] After filtration of the charcoal catalyst, cytochalasin C was isolated from any remaining cytochalasin D in the reaction product using
C-18 reverse phase thin layer chromatography plates with methanol:water, 75:25 v/v as mobile phase, followed by fluorescence quenching A small amount of commercial cytochalasin C (Sigma-Aldrich Corp.) was characterized
by reverse phase thin layer chromatography and recrystal-lized from acetone:hexane for comparison with the puri-fied product
Preparation of other cytochalasin congeners All other cytochalasin congeners (cytochalasins A, E, H,
J, and DiHCBγ-L), were acquired commercially (Sigma-Aldrich Corp.) All cytochalasins used in the study were
EtOH in conical 1.5 ml plastic centrifuge tubes Once the
10 mg/ml cytochalasin/EtOH solution was diluted into
EtOH
Preparation of clinically approved chemotherapeutic agents
Doxorubicin, paclitaxel, and vinblastine were acquired commercially (Sigma-Aldrich Corp.) Doxorubicin was solubilized in isotonic saline, paclitaxel in 1:1 100 % EtOH: Kolliphor EL, and vinblastine in sterilized water Plate assay procedures
SK human ovarian carcinoma cells were tested for drug sensitivity using 24-well assay plates (Corning Life Sci-ences, Corning, NY, USA) Each well contained ~ 1000 cells in 1 ml medium Agents were then dissolved in the wells at varying concentrations for 23 wells, while the last well remained untreated The plates were incubated at
37 °C for the length of drug exposure, and then stained with methylene blue This method was then used to assess varying inhibitory concentrations of each agent alone or in combination with another agent as described in [18] The
Trang 4efficacy of the method was also confirmed with a XTT
cells were seeded per well into a flat-bottom 96-well
microtiter plate in triplicate for each cell dilution The
plate was incubated for 24 h prior to addition of XTT
solution Cells were then incubated for an additional 2 h
before the wavelength was read
Examining the effects of cytochalasins on microfilaments
in neoplastic cells
Cytochalasins were assessed for their ability to inhibit the
formation of F-actin by examining the ratio of F-actin to
monomeric globular (G)-actin found within the SK human
ovarian cancer cell lines prior to and after treatment The
F-actin to G-actin ratio was determined with the G-Actin/
F-Actin In Vivo Assay Biochem Kit (Cytoskeleton Inc.,
Denver, CO, USA) After being treated, cells were lysed
with LAS2 buffer (1 ml lysis and F-actin stabilization buffer,
100× protease inhibitor cocktail stock solution) on ice for
10 min Cells were collected and the cell extracts were
centrifuged at 4 °C for 75 min at 16,000 g to separate the
F-actin and G-F-actin pools The supernatants of the extracts
were collected and designated as the G-actin pool The
pellets were resuspended in ice-cold actin depolymerization
buffer and designated as the F-actin pool Equal amounts of
both the supernatant (G-actin) and the resuspended pellet
(F-actin) were subjected to Western blot analysis with the
use of an anti-β-actin antibody
Assessing the inhibitory activity cytochalasins have
toward ATP binding cassette transporters
To determine whether cytochalasins exert antineoplastic
activity via inhibition of P-gp and other ABC transporters,
reverse transcription polymerase chain reaction (RT-PCR)
was used to quantify the RNA levels of three ABC
trans-porters; P-gp (ABCB1), Multidrug resistance-associated
protein 1 (MRP1; ABCC1) and multidrug
resistance-associated protein 2 (MRP2; ABCC2) Total RNA was
extracted from cells according to instructions provided in
the RNeasy Mini kit (Qiagen Inc., Valencia, CA, USA)
Suc-cessfully extracted RNA was then dissolved in
diethylpyro-carbonate/water Absorption values were read at 260 nm
and 280 nm using a UV spectrophotometer Acquired RNA
was converted into cDNA according to the instructions
provided in the RT-PCR kit (Life Technologies, Grand
Island, NY, USA), and primers used for RT-PCR are shown
in Table 1 The reaction was carried out under the
follow-ing conditions: denaturation at 95 °C for 5 min with an
additional 15 s at 94 °C, and a 30 s annealing at 60 °C
Targets genes were directly quantified to the reference gene
β-actin, since cytochalasins do not significantly affect the
RNA expression levels of actin [1]
In addition to examining the effects of cytochalasins on the RNA expression of ABC transporters in human ovarian carcinoma cells, direct inhibitory activity toward drug efflux pumps was assessed with rhodamine 123 (Rh123; 6-amino-9-(2- methoxycarbonylphenyl) xanthen-3-ylidene]azanium chloride) Cells were initially plated at 2 × 105cells/cm2in 24-well plates and allowed to reach 80 % confluence To assess the ability of cytochalasins to potentiate Rh123
presence and in the absence of cytochalasins or the known P-gp inhibitor verapamil for varying lengths of time At each time point, cells were collected, washed, resuspended
in ice-cold phosphate-buffered saline and kept on ice before fluorescence intensity was measured with flow cytometry Cells were also assessed for their ability to efflux accumu-lated Rh123 in the presence of cytochalasins or verapamil
30 min and then washing the cells twice with ice-cold PBS before being resuspended in fresh Rh123-free medium with and without agents Cells were collected at various time points and were then washed and resuspended in ice-cold PBS before being analyzed by flow cytometry
Determining the extent of drug synergy between cytochalasins, doxorubicin, and paclitaxel
To assess whether cytochalasin B or DiHCB synergizes with doxorubicin or paclitaxel against SK human ovarian carcinomas in vitro, cells were treated alone, or in
both the methylene blue and XTT assays In addition, the Chou-Talalay method for assessing drug synergism was implemented to determine the combination index (CI), dose reduction index (DRI), and fraction affected (Fa) As indicated in [27], synergism was assessed with the following values: CI < 1 (synergy) CI = 1 (additive)
CI > 1 (antagonism) In addition, DRI > 1 is representa-tive of favorable dose reduction, while DRI < 1 is repre-sentative of unfavorable dose reduction [27]
Results
Comparison of cytochalasin congeners and other antineoplastic agents against SK human ovarian carcinoma cell lines
There were notable differences in cytotoxicity between cytochalasin congeners and other antineoplastic agents against the human ovarian carcinoma cell lines Table 2
for nine cytochalasin congeners and for doxorubicin, paclitaxel, and vinblastine While doxorubicin, paclitaxel,
against the parent SKOV3 cell line after a 96 h expos-ure indicative of relatively high efficacy, (11 nM, 2.8
nM, and 1.9 nM, respectively), these agents had
Trang 5resistant SKVCR0.015 and SKVCR0.1 lines (125 nM and
125 nM for doxorubicin, 8 nM and 4 nM for paclitaxel,
and 10 nM and 20 nM for vinblastine, respectively),
indica-tive of drug resistance The SKVLB1 cell line showed even
higher resistance to these three agents exhibiting much
respectively; Table 2) The profound differences in
cytotox-icities of these agents against the drug sensitive parental
SKOV3 line compared to the multidrug resistant SKVLB1
line produced very high resistance indices (RI values)
These RI values ranged from 264 for doxorubicin to 1,400
and 1,600 for paclitaxel and vinblastine for SKVLB1 in
comparison with SKOV3
By contrast, cytochalasins did not show this marked
in-crease in resistance against the multidrug resistant SKVLB1
line (Table 2) In fact, cytochalasin A produced a lower IC90
value against SKVLB1 (750 nM) than against SKOV3
(1,000 nM) Interestingly, the most cytotoxic congeners
SKOV3 (cytochalasins C, D, E, and H) had higher RIs
against SKVLB1 than did cytochalasins A, B, J, DiHCB, or
initial cytotoxicity against SKOV3) Nevertheless, the
congener with the highest RI against SKVLB1, cytochalasin
C (RI = 100 against SKVLB1), still had a considerably lower
RI than the clinically approved chemotherapeutic agents (doxorubicin RI = 264, paclitaxel RI = 1,400, and vinblastine
RI = 1,600) The differences in cytotoxicity expressed as
IC90values along with the respective RI values as shown in Table 2 are further highlighted in Fig 2, which uses a loga-rithmic scale to compare the cytotoxicities of agents against SKOV3 and SKVLB1
Effects of a calcium ion channel blocker on cytochalasin-mediated cytotoxicity
As shown in Table 3, the calcium ion channel blocker verapamil increased the drug sensitivity of parental SKOV3
to cytochalasins A, B, and DiHCB by 1.2 to 2-fold Sensitiv-ity to cytochalasins C and D was not enhanced With respect to the highly drug-resistant SKVLB1 line, verapamil did not notably increase cytochalasin A cytotoxicity This presumably reflects the fact that cytochalasin A has slightly enhanced cytotoxicity for SKVLB1 than it does for SKOV3 Sensitivity of SKVLB1 to cytochalasin B or DiHCB was increased by 1.5 fold Very strikingly, the sensitiv-ity of SKVLB1 to cytochalasins C and D was markedly increased by 32- and 64-fold respectively The SK lines with intermediate resistance to SKVLB1 (SKVCR0.015 and SKVCR0.1) showed a 1.5 to 2-fold increase in sensitivity to cytochalasins A, B, and DiHCB when
Table 1 Primer sequences used for reverse transcription polymerase chain reaction to quantify RNA expression of ATP binding cassette transporters in SK human ovarian carcinoma cell lines
Table 2 Effects of clinically approved natural product antineoplastic agents and of cytochalasin congeners on drug-sensitive and multidrug-resistant human ovarian carcinoma lines
ND not determined, RI resistance index compared to parental SKOV3 line
a
Trang 6Fig 2 Comparison of clinically approved chemotherapeutic agents and cytochalasin congeners against SKOV3 drug sensitive and SKVLB1 multidrug resistant human ovarian carcinomas Abbreviations used are as follows: ADR (Adriamycin; doxorubicin), Tax (paclitaxel), VBL (vinblastine),
CA (cytochalasin A), CB (cytochalasin B), DiHCB (21,22-dihydrocytochalasin B), CC (cytochalasin C), CD (cytochalasin D), CE (cytochalasin E), CH (cytochalasin H), and CJ (cytochalasin J) The concentration of each agent in nM needed to produce an IC 90 value at 96 h for either cell line is represented on a logarithmic scale The resistance index (RI) of each compound against SKVLB1 is indicated in bold underneath the abbreviations
Table 3 Effects of verapamil on the sensitivity of human ovarian carcinoma cell lines to cytochalasin congeners
Cytochalasin A
-Cytochalasin B
21, 22-Dihydrocytochalasin B
Cytochalasin C
Cytochalasin D
ND not determined, FS fold sensitization
Trang 7administered in combination with 15 μM verapamil, and
cytochalasin D had a similar increase in sensitivity with
sensitivity of SKVCR0.1 to cytochalasin C by 2-fold, but did
not affect cytochalasin C with respect to SKVCR0.015 The
carcin-oma sensitivity to cytochalasins B, C, D and DiHCB are
fur-ther highlighted in Fig 3a Verapamil enhanced the
cytotoxicities of cytochalasin B and DiHCB in all four cell
lines tested (black and grey bars), and it enhanced the
cyto-toxicities of cytochalasins C and D against SKVCR0.1 by
1.5 to 2-fold (red and blue bars) The dramatic increases in
cytotoxicities of 32- to 64-fold noted above for
cytochala-sins C and D against the highly drug resistant SKVLB1 line
clearly apparent in Fig 3a
ver-apamil in a 96 h exposure is exhibited at multiple concen-trations of cytochalasin A against SKVCR0.015 (Fig 3b),
as well as with the less potent cytochalasin B against the same cell line (Fig 3c) After continuous exposure for
potential increase in cytotoxicity facilitated by verapamil is further highlighted in dose response growth curves of SKVCR0.015 cells after being treated alone with
a
Cytochalasins were administered alone or
in combination with 30 µM verapamil for 96 hours of continuous exposure.
Numbers in black refer to fold sensitization elicited by the addition of 30 µM verapamil
SKVCR0.015 human ovarian carcinoma with or without 30 µM verapamil after
continuous exposure for 96 hours.
SKVCR0.015 human ovarian carcinoma with or without 30 µM verapamil after
continuous exposure for 96 hours.
2.5 fold increase in sensitivity at 0.5 µM
2.8 fold increase in sensitivity at 0.25 µM.
Fig 3 Effects of verapamil and cytochalasins on SK human ovarian carcinoma cell lines a Effects of verapamil on potentiating SK human ovarian carcinoma sensitivity to cytochalasins B, C, D, and 21,22-dihydrocytochalasin B Cell lines are arranged in order of increasing drug resistance from left to right The IC 90 concentrations are given in μM Numbers in black refer to fold sensitization elicited by the addition of 30 μM verapamil.
b Effects of cytochalasin A-alone or in combination with 30 μM verapamil against SKVCR0.015 cells The values are taken as a percentage of
30 μM verapamil-treated cells c Effects of cytochalasin B-alone or in combination with 30 μM verapamil against SKVCR0.015 cells The values are taken as a percentage of 30 μM verapamil-treated cells d Dose response of cytochalasin A-alone or in combination with 30 μM verapamil on the growth of SKVCR0.015 cells e Dose response of cytochalasin B-alone or in combination with 30 μM verapamil on the growth of SKVCR0.015 cells For panels d and e, agents were administered for either 48 or 96 h, as indicated in the graphs
Trang 848 or 96 h While the addition of verapamil does not
con-siderably increase the percent growth inhibition at most
time points for cytochalasin A (Fig 3d), the calcium ion
channel blocker does potentiate cytochalasin B mediated
growth inhibition, particularly at lower concentrations
(Fig 3e)
Effects of cytochalasins and verapamil on the F/G-actin
ratio found in SK human ovarian carcinoma cells
As expected, cytochalasins demonstrated varying levels of
microfilament inhibition against the parental SKOV3 cells,
with the more potent agents potentiating lower F/G-actin
ratios (Fig 4) In agreement with their IC90 values against
SKOV3, cytochalasin D elicited the lowest F/G actin ratio
after 24 h (8.2), while DiHCBγ-L elicited the highest (20.9)
Interestingly, it appeared that 30μM verapamil had a small,
but notable effect on the F/G-actin ratio of SKOV3 cells
The efficacy of various cytochalasin congeners to inhibit
actin polymerization was notably different in multidrug
resistant SKVLB1 Although the cell line had a smaller, but
notable baseline F/G-actin ratio than its parental
counter-part (27.5 to 31), SKVLB1 was less sensitive to cytochalasin
D inhibition (15.9), but much more sensitive to cytochalasin
was indicated by the four day IC90concentrations The F/
G-actin ratio potentiated by cytochalasin B/verapamil was
lower than cytochalasin B-alone for both SKOV3 (13.4 to
14.3) and SKVLB1 (11.9 to 13.5) The notable decrease in
the F/G actin ratio elicited by the concomitant
administra-tion of cytochalasin B/verapamil follows the same pattern
observed in the cytotoxicity assays of Fig 3, suggesting
verapamil may have a slight, but notable influence on
cyto-chalasin B-mediated cytotoxicity
Assessment of ATP binding cassette transporter overexpression in SK human ovarian carcinomas and the inhibitory effects of cytochalasins and verapamil RT-PCR quantification of ABC transporters revealed that P-gp was substantially overexpressed in the drug resistant derivatives of SKOV3, with expression mirroring the level
of drug resistance associated with each cell line (Fig 5a) The overexpression of P-gp in SKVLB1 in comparison to parental SKOV3 is dramatic as Fig 5a required the use of a logarithmic scale to quantify all expression levels This pat-tern was also observed with MRP2, albeit at lower expres-sion levels, while MRP1 increased only minimally between the four cell lines RT-PCR also revealed that neither
influenced RNA expression, with a slight, but noticeable decrease in P-gp levels against all four neoplastic cell lines Nevertheless, cytochalasins A and B appeared to have notable inhibitory activity against ABC transporters, as assessed by Rh123 accumulation and efflux analysis
cytocha-lasin A or B readily accumulated Rh123, becoming satu-rated by the dye at 60 min (Fig 5b) This activity was also
C, which had only a slight influence on accumulation levels All treatment groups for SKOV3 became saturated
by Rh123, indicative of its low expression of P-gp and other efflux pumps The activity of cytochalasins A and B,
as well as verapamil against ABC transporter-mediated ef-flux of Rh123 was confirmed by experiments in which cells were incubated with the dye prior to being placed in
cytochala-sin C treated SKVLB1 cells effluxed Rh123 at a consistent
Fig 4 Effects of cytochalasin congeners on the F/G-actin ratio of SKOV3 and SKVLB1 human ovarian carcinomas Abbreviations are the same as those used in Fig 2, except for the addition of Ve (verapamil) All cytochalasins were administered at their IC 90 value of 96 h continual exposure, and Ve was administered at 30 μM The F/G-actin ratio of both SKOV3 and SKVLB1 cells was assessed 24 h post-administration Bars represent standard error of the mean (SEM) for each treatment group
Trang 9rate with untreated cells reaching 12.4 % content of the
original Rh123 incubation and cytochalasin C treated cells
A or B retained much higher percentages of accumulation
(62.4 % and 53.1 %, respectively), but were less effective
Efficacy of cytochalasin B in increasing drug sensitivity
Cytochalasin B appeared to increase the drug sensitivity of
SKVLB1 cells to clinically approved antineoplastic agents
known to have reduced cytotoxicity against this multidrug
resistant cell line (Table 4) The IC values of doxorubicin
against SKVLB1 cells at 13.5 h and at 33 h of exposure and for paclitaxel at 13.5 h dropped by 2 to 4-fold using
It should be noted that much higher concentrations of
values against SKVLB1 than in earlier determinations because the earlier values were measured at 96 h of expos-ure, while the combination of cytochalasin B with doxo-rubicin or paclitaxel were measured at 13.5 and 33 h
Fig 5 Effects of cytochalasin congeners and verapamil on RNA expression of ATP binding cassette proteins and the efflux of rhodamine 123 in
SK human ovarian carcinoma cell lines a Relative RNA expression of ABCB1 (P-gp), ABCC1 (MRP1), and ABCC2 (MRP2) in SK human ovarian carcinoma cell lines prior to and after treatment with cytochlalasins as assessed by RT-PCR Primer sequences used in the reaction are shown in Table 1 b Cells were treated with the indicated agents, and then exposed to Rh123 c Cells were incubated with Rh123, and then placed in fresh medium The concentrations of each agent used are indicated in the individual panels Bars represent SEM for each treatment group
Trang 10Assessment of drug synergy between cytochalasins and clinically approved agents
Both cytochalasin B and DiHCB appeared to synergize with doxorubicin and paclitaxel in SKOV3 and SKVLB1 cells
isobolo-grams (Fig 6) The synergy indicated by these values was also confirmed with Chou-Talalay CI values (Table 5) In addition, DRI values for both the cytochalasins and cur-rently approved agents were indicative of favorable dose reductions, although this would be expected from the isobolograms Interestingly, in both measurements of syn-ergy, it appeared that cytochalasin B and DiHCB synergized with the clinically approved agents more strongly against SKVLB1 than against drug sensitive SKOV3 This syner-gism was noted at other inhibitory concentrations, as indi-cated in Fig 7 Although the individual values may vary, the lines of the Fa-CI plot for SKVLB1 appear to be noticeably
Table 4 Sensitivity to clinically approved antineoplastic agents
potentiated by cytochalasin B against multidrug resistant
SKVLB1 human ovarian carcinoma
Doxorubicin
Paclitaxel
FS fold sensitization
Fig 6 IC 50 isobolograms for cytochalasin B, 21, 22-dihydrocytochalasin B, doxorubicin, and paclitaxel against SKOV3 and SKVLB1 human ovarian carcinoma a CB and ADR b CB and Tax c DiHCB and ADR d DiHCB and Tax IC 50 values were determined after a 48 h continuous exposure